AU659486B2 - Novel alpha-mannosidase inhibitors - Google Patents

Abstract

[4S-(4 alpha ,4a beta ,5 beta ,6 alpha ,7 alpha ,7a alpha )]-Octahydro-1H-1-pyrindine-4,5,6,7-tetrols and [4R-(4 alpha ,4a alpha ,5 alpha ,6 beta ,7 beta ,7a beta )]-octahydro-1H-1-pyrindine-4,5,6,7-tetrols are useful as inhibitors of alpha-mannosidase and are useful immunostimulants, chemoprotective and radioprotective agents and antimetastatic agents.

Description

OPI DATE 27/04/93 AOJP DATE 24/06/93 APPLN. ID 24771/92 PCT NUMBER PCT/US92/06707 llll ll llll i ll111111111liii AU9224771 IN I ZKNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 93/05781 A61K 31/44 Al (43) International Publication Date: 1 April 1993 (01.04.93) (21) International Application Number: PCT/US92/06707 (74) Agent: NESBITT, Stephen, Merrell Dow Pharmaceuticals Inc., 2110 East Galbraith Road, P.O. Box 156300, (22) International Filing Date: 10 August 1992 (10.08.92) Cincinnati, OH 45215-6300 (US).

Priority data: (81) Designated States: AU, CA, FI, HU, JP, KR, NO, Euro- 761,579 18 September 1991 (18.09.91) US pean patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, 893,171 3 June 1992 (03.06.92) US IE, IT, LU, MC, NL, SE).

(71)Applicant: MERRELL DOW PHARMACEUTICALS Published INC. [US/US]; 2110 East Galbraith Road, P.O. Box With international search report.

156300, Cincinnati, OH 45215-6300 (US).

(72) Inventors: FARR, Robert, A. 2960 Maureen Ct., Love- 8 land, OH 45140 KANG, Mohinder, S. 4476 Edenton Lane, Cincinnati, OH 45242 PEET, Norton, 8028 Chestershire Drive, Cincinnati, OH 45241 (US).

SUNKARA, Sai, P. 9361 Bluewing Terrace, Cincinnati, OH 45236 (US).

(54)Title: NOVEL ALPHA-MANNOSIDASE INHIBITORS (57) Abstract [4S-(4u, 4ap, 51, 6a, 7a, 7au)]-Octahydro-lH-l-pyrihdine-4,5,6,7-tetrols and [4R-(4a, 4aa, 5a, 6p, 713, 7ap)]-octahydro-IH- 1-pyrindine-4,5,6,7-tetrols are useful as inhibitors of alpha-mannosidase and are useful immunostimulants, chemoprotective and radioprotective agents and antimetastatic agents.

~ILII LL I i"

I

'I

i i1 11

I

i; la WO 93/05781 PCT/US92/06707 -1- NOVEL ALPHA-MANNOSIDASE INHIBITORS' BACKGROUND OF THE INVENTION application serial num ~7 Tj -i ied September 18, 10 Most human cancers are treated by the use of radio and/or chemotherapeutic agents. There are mainly two problems associated with the use of such techniques in the treatment of cancer, namely, adverse side effects and resistance to the use of cytotoxic chemical agents. .These phenomena limit the usefulness of such therapies. Regulation of the growth and differentiation of hematopoietic progenitor cells is under the control of several growth factors. It is well known that immunomodulators such as interleukins, granulocyte-macrophage colony stimulating factors, tumor necrosis factor and bacterial lipopolysaccharides, due to their ability to enhance hematopoietic and immune functions, confer radio and chemoprotection.

Recently, swainsonine has been demonstrated to mimic growth promoting activities of various hematopoietic growth factors and offer protection from radiation and cytotoxic chemotherapy. S. L. White et al., Cancer Communications, 83-91, 1991.

WO 93/05781 PCT/US92/06707 -2- Swainsonine, a plant alkaloid initially isolated from Australian plant Swainsona canescens, has been demonstrated to be a potent and specific inhibitor of golgi alphamannosidase II, a'glycoprotein processing enzyme.

(Colegate et al., Aust. J. Chem. 32: 2257 2264, 1979.) Swainsonine has also been shown to have many other biological effects including inhibition of tumor growth, metastasis, augumentation of host immune effector mechanisms and activation of protein kinase-C. Kino et al., J. Antibiotics, 38(7), 926 940, 1985.) Although it is not clear by which mechanism(s) swainsonine offers radio/chemoprotection, inhibitors of alpha-mannosidase and analogs of swainsonine are expected to be useful as radio/chemoprotective agents.

Applicants have now discovered a new class of alphamannosidase inhibitors which are useful as immunomodulators, chemo- and radioprotective agents and as antimetastatic agents.

SUMMARY OF THE INVENTION This invention relates to'novel alpha-mannosida3e inhibitors of formula 1

OH

HO' 3 HO- 3 7

HO

R

IICYI~--L 1311~-. 1 I ~I C-7 I WO 93/05781 PCT/US92/06707 -3wherein R is a hydrogen, a (C 1

-C

6 )alkyl optionally substituted with one or two hydroxy groups, a glycosyl group, or a group of the formula

-(CH

2 )n-Ar wherein n is an integer of from 1 to 4 and Ar is a phenyl group optionally substituted with one or two groups selected from (Ci-C 4 )alkyl,

(C

1

-C

4 )alkoxy, F, Cl, Br, I, amino, mono(C 1

C

4 )alkylamino, or di(C 1

-C

4 )alkylamino, or a pharmaceutically acceptable salt thereof, whi~te C> presetQ r^a-1 \Cde useful as immunostimulants, chemoprotective agents, radioprotective agents and as antimetastatic agents.

DETAILED DESCRIPTION OF THE INVENTION The usual stereochemical conventions are used throughout to denote the relative spatial orientation of groups attached to the rings. Thus, a solid line diverging from the point of attachment to a ring indicates that the Sattached group is in the beta-configuration, that is, the group is above the plane of the ring. Likewise, a dotted line indicates that the attached group is in the alphaconfiguration, that is, the group is below the plane of the ring. Attachment of a group to a ring by a normal, not 2 divergent or dotted, line indicates that the spatial orientation can be either alpha or beta.

The (C 1

-C

6 )alkyl groups of this invention can be straight chained, branched chain or cyclic. Examples of 3 such alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, Scyclopentyl, n-hexyl, and cyclohexyl.

In those alkyl groups substituted with two hydroxy groups, the hydroxy groups will not be bonded to the same

A-

,,U1 .L I I I WO 93/05781 PCT/US92/06707 carbon atom. Further, the hydroxy group will not be bonded to the carbon atom which is bonded to the amino nitrogen atom.

The glycosyl groups of this invention can be mono-, dior trisaccharide moieties. The glycosyl group can be attached to the amino nitrogen atom through either an exocyclic or ring carbon atom of the glycosyl pentose or hexose ring thereby forming a variety of possible positional isomers for each individual glycosyl group.

Also similar or dissimilar pentose or hexose moieties may be linked to each other through a glycosidic oxygen bridge wherein the bridging oxygen atom is attached to an exocyclic and/or endocyclic carbon atom of the pentose or hexose moiety of which the glycosyl radical is comprised; again all positional isomers are contemplated as within the scope of this invention.

Exemplary of glycosyl radicals contemplated are .such monosaccharides as glucosyl, galactosyl, mannosyl, fucosyl, ribosyl, 2-deoxyglucosyl, 3-O-methylglucosyl, xylosyl, and arabinosyl, disaccharides as alpha- and beta-cellobiosyl, isomaltosyl, trehalosyl, and maltosyl, and such trisaccharides as maltotriosyl, and cellotriosyl. Particularly preferred are the compounds wherein R is mannosyl.

Acid addition salts with pharmaceutically acceptable acids referred to above are equivalent to the amines for the purposes of this invention. Illustrative of such salts are the salts with inorganic acids such as, for example, hydrochloric, hydrobromic, sulfuric, phosphoric and like acids; with organic carboxylic acids such as, for example, acetic, propionic, glycolic, lactic, pyruvic, malonic, succinic, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic and dihydroxymaleic, benzoic,

I

~IL~3C WO 93/05781 PCT/US92/06707 phenylacetic, 4-aminobenzoic, 4-hydroxybenzoic, anthranilic, cinnamic, salicylic, 4-aminosalicylic, 2phenoxybenzoic, 2-acetoxybenzoic, mandelic and like acids; and with organic sulfonic acids such as methanesulfonic acid and p-toluenesulfonic acid. Such salts can be obtained by standard,procedures from an amine of this invention and the appropriate acid.

Of those compounds of formula 1, those compounds wherein R is a methyl or ethyl, benzyl, a 1,3dihydroxyprop-2-yl, 2-hydroxypropyl and mannosyl are preferred. Also preferred are those compounds of formula 1 whererin the 4-hydroxy group is in the alpha-configuration.

The compounds of formula 1 can be prepared by techniques and procedures well known and appreciated by one of ordinary skill in the art. A general synthetic scheme for preparing the compounds of this invention is set forth in Scheme A. In Scheme A, all subsLitutents are as previously defined unless otherwise indicated.

WO 9305781PCT/US92/06707 WO 93/05781 -6- Scheme A B nO, B no~- BnO

CHO

BnO step a

M

0

PMB

~OH

PMB

step c step b B nO

~\CHO

'-Boc

PMB

B nO, ,,oCHO step d B ./'N-Boc

PMB

OH

step e step f

PMB

PCr/US92/06707 WO 93/05781 B n'0, B nON-~ B nO

B

BnOi -7- Scheme A Cont.

'N-Boc C0 2 Et BnO B Bno step g

OH

2 step h stepi 1

H

B nO BnOlf- 10a)

H

(lob) H PCT/US92/06707 WO 93/05781 -8- Scheme A Cont.

BnO! BnOba- Bn riO 1a)

H

(10b) H optionial step jl optional Sstep j2 (11a) Sstep k 1 (1ib) Sstep k2 HO

HO

(12a)

R

(12b) R RI other than hydrogen WO 93/05781 PCfIUS92/06707 -9- Scheme A provides a general synthetic.procedure for preparing compounds of formula 1.

In step a, 2,3, 4-tris (phenylmet ,hoxy)-5 ,6-dideoxy-Dlyxo-hex-5-enose is reacted with 4-methoxybenzylhydroxylamine to give [3aS-(3aca,46,5a,6a,6aa) ]-hexaiiydro-l- [(4-methoxyphenyl)methyl)-4,5,6-tris(phenylmethoxy -lHcyclo-pent fjc]isoxazole For example, 2,3,4-t'ris(phenylmethoxy)-5,6-dideoxv-Dis contacted with an slight molar excess of 4-methoxybenzylhydroxylamine. The reactants are typically contacted in a suitable protic organic solvent such as methanol. The reactants are typically contacted for a period of time ranging~ from 5-24 hours at a temperature range of from room. temperature to reflux.

[3aS-(3act,4B,5a,6a,6aa) ]-Hexahydro-l-[ 4-methoxyphenyl)methyl]-4,5,6-tris(phenylmethoxy)-lHi-cyclopent[clisoxazcl~e is recovered from the reaction zone by basification followed by extractive methods as is known in the art. It may be purified by silica gel chromatography.

In step b, (3aS-(3aa,4B,5a,ca,6aa)]-hexahydro-l-((4methoxyphenyl)methylj-4,5,6-tris(phenylmethoxy)-lHcyclopent[c]i~oxazole is reduced to give [lS-(la,2a,3a,4B,5S) ]-[2-(hydroxyraethyl)-3,4,5 (phenylmethoxy)cyclopentyl] .nethoxyphenvl)methyl ]amine followed by acylation with di-tert-but yl dica rbonate to give [lS-(la,2a,3a,'4B,5B) I-2-(hydroxymethyl)-3,4,5- (phenylmethoxy)cyclopentyl (4-methoxyphenyl)methyl 3carbamic acid, 1,1-dimethylethyl ester The reduction can be accomplished by any means known to.

those skilled in the art f.or r-eduction of the oxygennitrogen bond provided that the reaction conditions do not i WO 93/05781 PC/US92/06707 substantially affect the relative stereochemistry of the groups.

For example, [3aS-(3aa,4B,5a,6a,6aa)]-hexahydro-l-[(4methoxyphenyl)methyl]-4,5,6-tris(phenylmethoxy)-lHcyclopent[c]isoxazole of structure is contacted with a molar excess of activated zinc dust. The reactants are typically contacted in a suitable acid medium such as acetic acid. The reactants are typically stirred together for a period of time ranging from 1-5 hours at a temperature range of from room temperature to reflux. The intermediate [lS-(la,2a,3a,4B,5 i-[2-(hydroxymethyl)- 3,4,5-(phenylmethoxy)cyclopentyl)[(4-methoxyphenyl)methyllamine is recovered from the reaction zone by extractive methods as is known in the art.

The intermediate [1S-(la,2a,3a,4B,5B)]-[2- (hydroxymethyl)-3,4,5-(phenylmethoxy)cyclopentyl][(4methoxyphenyl)methyl]amine is contacted with a molar excess of di-tert-butyl dicarbonate. The reactants are typically contacted in a suitable aprotic organic solvent such as tetrahydrofuran. The reactants are typically stirred together for a period of time ranging from 2-24 hours at a temperature range of from room temperature to reflux. [lS- (la,2a,3a,4B,58))-[2-(Hydroxymethyl)-3,4,5- (phenylmethoxy)cyclopentyll[(4-methoxyphenyl)methyl]carbamic acid, 1,1-dimethylethyl ester is recovered from the reaction zone by extractive methods as is known in the art. It may be purified by lica gel chromatography.

In step c, [1S-(la,2a,3a,48,58)]-2-(hydroxymethyl)- 3,4,5-(phenylmethoxy)cyclopentyll[(4-methoxyphenyl)methyl}carbamic acid, 1,1-dimethylethyl ester is oxidized, to give [lS-(la,2a,3a,43,58)5-[2-formyl-,4,5- WO 93/05781 PCr/US92/06707 -11tris(phenylmethoxy)cyclopentyll[(4-methoxyphenyl)methyl]carbamic acid, 1,1-dimethylethyl ester The oxidation can be accomplished by any means known to those skilled in the art for oxidation of the hydroxymethyl group provided that the reaction conditions do not substantially affect the relative stereochemistry of the other positions, or cause one of the substituents on the cyclopentane ring to be lost in a B-elimination process.

For example, [1S-(la,2a,3a,4B,56))-[2-(hydroxymethyl)- 3,4,5-(phenylmethoxy)cyclopentyll[(4-methoxyphenyl)methyl]carbamic acid, 1,1-dimethylethyl ester is contacted with 2 molar equivalents of Dess-Martin periodinane. The reactants are typically contacted in a suitable aprotic organic solvent such as methylene chloride. The reactants are typically stirred together for a period of time ranging from 5 minutes to 2 hours and at a temperature range of from 0 0 C to room temperature. [lS-(la,2a,3a,4B,5 Formyl-3,4,5-tris(phenylmethoxy)cyclopentyl]l(4methoxyphenyl)methyl]carbamic acid, 1,1-dimethylethyl ester is recovered from the reaction zone by extractive methods as is known in the art.

Alternativel-, [lS-(la,2a,3a,48,58)I-[2-(hydroxymethyl)-3,4,5-(phenylmethoxy)cyclopentyl][(4-methoxyphenyl)methyl]carbamic acid, 1,1-dimethylethyl ester is contacted with a molar excess of dicycloexyl-carbodiimide, molar excess of dimethylsulfoxide, a slight molar excess of pyridine and a molar deficiency of an acid such as trifluoroacetic acid. The reactants are typically contacted in a suitable aprotic organic solvent such as toluene. The reactants are typically stirred together for a period of time ranging from 1-24 hours and at a temperature range of from 10bC to room temperature. [lS- WO 93/05781 PCr/US92/067 07 (la,2a,3a,4B,5B)]-[2-Formyl-3,4,5-tris(phenylmethoxy)cyclopentyl] [(4-methoxyphenyl)methyllcarbamic acid, 1,1dimethylethyl ester(4) is recove'red from the reaction zone by extractive methods as is known in the art.

In step d, the alpha carboxaldehyde of cyclopentyl] [(4-methoxyphenvl)methyllcarbamic acid, 1,1dimethylethyl ester is inverted to the beta position to j 10 give [1S-(lc ,2B,3a,4R,5B) -[24-forzyl-3,4,5-tris(phenylmethoxy)cyclopentyl] [(4-methoxyphenyl)methyl]-carbamic acid, 1,1-dimethylethyl ester For example, [lS-(la,2c,3a,4B,5B)]-[2-formyl-3,4,5tris(phenylmethoxy)cyclopenty-] (4-methoxyphenyl)methyl]carb~mic acid, 1,1-dimethylethyl ester is contacted with less than a molar excess of a non-nucleophilic base such as l,8-diazabicyclo[5.4.0]undec-7-ene. The reactants are typically contacted in a suitable aprotic organic solvent such as methylene chloride. The reactants are A typically stirred together for a period of time ranging from 1-5 hours and at a temperature range of from -78 0 C to tris(phenylmethoxy)-cyclopentyl] (4methoxyphenyl)methyllcarbamic acid, 21 l-d imethylethyl ester is recovered from th, reaction zone by acidification followed by extractive methods as is known in the art.

Alternatively, the alpha carboxaldehyde of I[lS-(la,2ci,3a,4B,5B) )-[2-formyl-3,4,5-tris(phenylmethoxy)cyclopentyl] [(4-methoxyphenyl)methyllcarbamic acid, 1,2.dimethylethyl ester is inverted to the beta position to give [lS-(la,28,3a,4B,5.8)]-t2-formyl-3,4,5-tris(phenylmat-hoxy)cyclopentyl] C(4-methoxyphenyl)methyllcarbamic acid, WO 93/05781 PCT/US92/06707 -13- 1,1-dimethylethyl ester merely by silica gel chromatography.

In step e, [lS-(laL,2B,3a,4B,5B)]-[2-formyl-3,4,5tris(phenylmethoxy)cyclopenty'L] [(4-methoxyphenyl)methyljcarbamic acid, 1,1-dimethylethyl ester is alkylated with ethyl acetate to give [lR,2S,3R,4S,5R]-'2-[[(1,1dimethylethoxy)carbonyl] [(4-methoxyphenyl)methylIlamino]-Bhydroxy-3 ,4 ,5-tris (phenylmethoxy )-cyclopen'tanepropanoic acid, ethyl ester For example, a molar excess of ethyl acetate is first contacted with a suitable non-ntucleoohilic base such as lithium hexamethyldisilazide. The reactants are typically contacted in a suitable aprotic organic solvent such as tetrahydrofuran. The reactants are typically stirred together for a period of time ranging from 5-60 minutes and at a temperature range of from -78 0 C to -40 0

C.

A solution of [lS-(la.,2$,3a,4B,5B)]-[2-formyl-3,4,5tris(phenylmethoxy)cyclopentyl) t(4-xethoxyphenyl)methyl]carbamic acid, 1,1-diniethylethyl ester in a suitable aprotic organic solvent such as tetrahydrofuran is then added. The reactants are typically stirred together for a period of time ranging from 15 minutes to 1 hour and at a temperature range of from -78 0 C to -400C. [lR,2S,3R,4S,5R]- (l,-Dimethylethoxy)carbonyl] [(4-methoxyphenyl)methyl) amino I-B-hydroxy.-3, 4,5-tr is (phenylmethoxy) cyc lope ntanepr opano ic acid, ethyl1 ester i.s recovered from the reaction zone by acidification followed by extractive methods as is known in the art. It may be purified by silica gel chromatography.

In step f, the N-p-methoxybenzyl group of 35[lR,2S,3R,4S,5R]-2-[[(l,1-dimethylethoxy)carbonyllt(4- WO 93/05781 PCT/US92/06707 -21.4methoxyphenyl.)methyl lamino] -B-hydroxy-3, 4, 5-tris (phenylrethoxy)cyclopentanepropanoic acid, ethyl ester of structure (6)Yis removed to give [lR,2S,3R,4S,5R)-Bhydroxy-2-[ (,l-dimetL-hylethoxy)carbonyllamino1-3,4,5tris(phenylrnethoxy)cyclopentanepropanoic acid, ethyl ester For example, [lR,2S,3R,4S,5R1-2-[[(l,ldimethylethoxy)carbonyl (4-methoxyphenyl)methyllanino]-Bhydroxy-3,4,5-tris(phenylmethoxy)cyclopentanepropanoic acid, ethyl ester of structure is contacted with 3 molar equivalents of ceric ammonium nitrate. The reactants are typically contacted in a suitable protic solvent mixture such as acetonitrile/water. The reactants are typically stirred together for a period of 15 minutes to 3 hours and at a temperature range of from 0 0 C to room temperature. [lR,2S,3R,4S,5R]>2-[[(l,ldimethylethoxy)carbonyllamino]-s-hydroxy-3,4,5tris(phenylmethoxy)cyclopentaneprop.3noic acid, ethyl ester is recovered from the reaction zone by extractive methods as is known in the art. It may be purified by distillation and/or recrystallization.

in step g, the N-tert-butylcarboxy group of [lR,2S,3R,4S,5R]-2-E[(l,1-dimethylethoxy)carbonyllamino]-Bhydroxy-3,4,5-tris(phenylmethoxy)cyclopentanepropanoic acid, ethyl ester is removed to give [lR,2S,311,4S,5F])- 2-amino-$-hydroxy-3,,4,5-tris(phenylmethoxy)cyclopentanepropanoic acid, ethyl ester For example, [lR,2S,3R,4S,5R1-2 dimethyleth'xy)carbonyllamino]-B-hydroxy-3,4 IStris(phen Ln(,thoxy)cyclopentanep~ropanoic acid, ethyl ester is cc-ntacted with a molar excess of hydrog'en chloride gas. The reactants are typically contacted in suitable .1 I b~M ~9~ WO 93/05781 PCI/US92/06707 aprotic organic solvent such as ethyl ether. The reactants are typically stirred together for a period of time ranging from 25-75 minutes and at a temperature range of from 0 0

C

to room temperature. [lR,2S,3R,4S,5R1-2-amino---Hydroxy- 3,4,5-tris(phanylmethoxy)cyclopentanepropanoic acid, ethyl ester as a mixture of epimers is recovered from the reaction zone by basification followed by extractive methods as is known in the art.

In step h, [lR,2S,3R,4S,5R])-2-amino-B-Hydroxy-3,4,5tris(phenylmethoxy)-cyc.opentanepropanoic acid, ethyl ester is cyclized to give [4aR,5R,6S,7R,7aS]-octahydro-4hydroxy-5,6,7-tris(phenylmethoxy)-2-l-pyrindif>oie For example, tlR,2S,3R,4S,5R]-2-amino--hydroxy-3,.4,5tris(phenylmethoxy)cyclopentanepropanoic acid, ethyl ester is contacted with a catalytic amount of a suitable base such as sodium methoxide in methanol. The reactants are tyically stirred together for a period of time ranging from 15 minutes to 5 hours and at a temperature range of from room temperature to reflux. [4aR,5R,6S,7R,7aS]-Octahydro- 4-hydroxy-5,6,7-tris(phenylmethoxy)-2H-lpyrindin>one (9) is recovered from the reaction zone by extractive methods as-is known in the art.

In step i, [4aR,5R,6S,7R,7aS3-octahyro-4-hydroxy- 5,6,7-tris(phenylme.thoxy)-2H-l-pyrindin-2-one is reduced to give [4S-(4a,4aB,5$,6a,7a,7aa)]-octahydro-5,6,Th tris(phenylmethoxy)-l-l-pyrindin-4-o1 of structure and [4R-(4a,4aa,5a,6 ]7B,7aB1 -dctahydro-5,6,7tris(phenylmethoxy)-lH--pyrindin-4-o1 For example, [4aR,5R,6S,7R,7aS]--octahydro-4-hydroxy- 5,6,7-tris(phenylmethoxy)-2H-l-pyrindin-2-one is contacted with a slight molar excess of a suitable reducing WO 93/05781 PCT/US92/06707 agent such as lithium aluminum hydride. The reactants are typically contacted in a suitable aprotic organic solvent such as tetrahydrofuran. The reactants are typically stirred together for a period of time ranging from hours land at a temperature range of from room temperature to reflux. [4S-(4ca,4aB,5 36a, 7a, 7aa) I -octahydro-5, 6,7tris(phenylmethoxy)-lH-a i -pyrindin-4-ol of structure and (4R-(4a,4aa,5cx,6B,7S,7aB) ]-octahy'dro-5,6,7tris(pheny'Lmethoxy)-1H--l-pyrindifl-4-ol (l0b) are recovered from the reaction zone by extractive methods as is known in the art and sep~arated by silica gel chromatography.

In optional step jj, [4S-(4cx,4aB,5B,6a,7a,7aa)] octahydro-5,6,7-tris(phenylmethoxy)-lH-l-pyrindin- 4 -oi of structure (10a) is N-alkylated to give the appropriate [4S- (4a,4aB,56, 6a,7a,7aa) ]-octahydro-l-alkyl-5,6,7tris(phenylmethioxy)-lH-l-py.rindin-4-ol of structure (11a).

For example, reductive amination of [4S- (4a,4aB,5f,6a,7a,7aa)J-octahydro-5,6,7-.tris(phenylmethoxy)lE-l-pyrindin-4-ol (10a) with an-equimolar amount of the appropriate aldehyde and an equimolar amount of sodium cyanoborohydride as is known in the art gives the appropriate .[4S-(4c,4aB,5 ,6a,7a,7aa) ]-octahydro-l-alkyl- 5,6,7-tris(phenylmethoxy)-l-l-pyrindin-4-ol of structure (11a).

Alternatively, alkylation of [4S- (4a,4aB,5$,6a,7a,7aa)J-octahydro-5,6,7-tris(phenylmethoxy)lH-l-pyrindin-4-ol (10a) with an equimolar amount of an apropriate alkyl halide and an equimolar amount of a suitable base such as potassium carbonate as is known in the art gives tae appropriate S a a,5,6,7,7a octahy dro-l-alkyl-5,6,7-tris (phenymethoxy) -HlHpyrildin- 4-ol of structure (11a).

'4 WO 93/05781 PCI! US92/06707 -17- In optional step j2, [4R-(4cx,4aa,5a,65,7B,7a octahydro-5,6,7-tris(phenylmethoxy)-l-1-pyrindin-4-ol (l0b) is N-alkylated to give [4-4,aL5a6,B7B3 octahydro-I---alkyl-5,6,7-tris(phenylmeth-oxy)-lH-l-pyrindin- 4-o. of structure (11b) as described in optional step ji.

In step [4S-(4ar,4a,5B,6a,7ca,7ac)]-octahydro-5,6,7tris(phenylmethoxy)-lH-l-pyrindin-4-ol (10a) or the appropriate [4S-(4a,4aB,5B,6a,7a,7aci)]-octahydro-l-alkyl- 5,6,7-tris(phenylmethoxy)-lH-l-pyrindin-4-ol of structure (11a) is deprotected to give the appropriate [4S- (4a,4aB,5B,6a,7o.,7aaj]-octahydro-lH-l-pyrindine--4,5,6,7tetrol of structure (12a).

For example, t4S-(4cL,4aB,5B,6lr7,7ax) ]-octahydro- 5,6,7-tris(phenylmethoxy)-lH-l--pyrindin-4-ol (10a) or [4S- (4c,4a,5B,6a,7a,7aca)-octahydro-l-alkyl-5,6,7tris(phenylmethoxy)-lH-1-pyrindin-4-ol of structure (11a) is contacted with hydrogen gas and a catalytic amount of palladium black. The reactants are typically contacted in a suitable acidic medium-such as acetic acid or methanolic hydrochloric acid. The reactants are typically shaken on a Parr hydrogenation apparatus at room tempeature for a period of time ranging from 2 hours to 10 days. The appropriate [4S-(4a,4aa,5a,6a,7a,7aca)]-octahydro-lH-lpyrindine-4,5,6,7-tetrol structure (12a) is recovered from the reaction zone by filtration and purified by ion exchange chromatography.

In step k 2 £4R-(4a,4axx,5a 6B,75,7aB) )-octahydro-5,6,7tris(phenylmethoxy)-IH-l-pyrindin-4-ol of structure or the appropriate t4R-(4a,4aa,5a,6B,7B,7aB.))-octahydro-lalkyl-5,6,1-tris(phenylmethoxy)-lH-1-pyrindin-4-ol ofI structure (11b) is deprotected to give the appropriate (4Rmaleic, hydroxymaleic, and dihydroxymaleic, benzoic, LP4 WO 93/05781 Pcr/US92/067b7 (4a,4aa,5a,6B,7B,7aB) ]-octahydro-IlH-l-pyrindine-4,5,6,7tetrol of structure (l2b) as described in step kj.

Starting materials for use in S cheme A are readily available to one of ordinary skill in the art. For example, the preparation of methyl-2,3,4-tris(phenylmethoxy)-6-bromo-6-desoxy-a-D-mannopyranose and its conversion to 2,3,4-tris(phenylmethoxy)-5,6-dideoxy-D-lyxoare described in Helv. Chim.Acta 62 .2400 1979.

The foDllowing examples present typical syntheses as described in Scheme A. These examples are understood to be illustrative only and are not intented to limit the scope of the present invention in any way. As used herein, th.

following terms have the indicated meanings: "q refers to grams; "mmol" refers to millimoles; "mL" refers to milliliters; "bp" refers to boiling point; 0 C" refers to degrees Celsius;."mm Hg" refers to millimeters of mercury; UjL" refers to microliters; "jig" refers to micrograms; and "pWM refers to micromolar.

Example 1 [4R-(4a,4aoa5a,6B,7B,7aB)]-Octahvdro-lH-1-ovrindine- 4,5,6, 7-tetrolahvdrochloride--MDL-l00 ,337A

OH

HO 0 HO~\ oHCI HO I

H

Step a: E3aS-(3aa,48,5a,6a,6aa)1-Hexahydro-l-[(4me thoxvDhen 1 me t hyl 1- 45, 6ztr is (Dheny lme thoxy) 1HcNyclopent~clisoxazclte i ~,WO 9j/05731 PCriUS92/06707 -19- Dissolve freshly distilled 4-methoxybenzaldehyde (45.56g, 334.6mxnol) in methanol (l75mL) and add hydroxylamine hydrochloride (30.06g, 0.433mo1) and stir for 15 minutes.

Add sodium methoxide (11.0g, 0.204mo1) and stir at room temperature for 1 hour, partially conceitrate invaciuo and add water (400mL). Extract into ethyl ether wash with saturated sodium chloride and dry (MgS0 4 Evaporate the solvent inuvacuo to give 4-methoxybenzyloxime as an off white solid (49.95g, 98.8%).

Dissolve 4-methoxybenzyloxime (1.49g, 9.B6mmol) in acetic acid (l5mL) and add sodium cyanoborohydride (0.937g, 14.9mmol). Stir at room temperature for 15 minutes, basify with aqueous potassium hydroxide and extract into ethyl ether. Wash with water (2X) dry (MgS0 4 and evaporate the solvent- invacuo to give 4-methoxybenzylhydroxylamine as a white solid (0.724g).

ixmethyl-2,3,4-tris(phenylmethoxy)6-bromo6desoxy-a-Dmannopyranose (20.968g, 39.7Smmol) and activated zinc dust (20.50g, 3l4rnmol) in isopropanol (300mL) and water Heat at reflux for 38 minutes, cool and filter. Rinse the zinc dust with a mixture of ethyl acetate/water, filter- and dilute th-itae with water.- Extract with a mixture of ethyl acetate/cyclohexane (3X) and wash the combined organic extracts with water. Evap:orate the solvent invacuo jto give 2,3,4--tris(phenylmethoxy)5,6dideoxy-.lyxo-hex-5enose.

Mix 2,3,4-tris(phenylmethoxy)-5,6-dideoxy-!-lyxr)hex-5enose (39.75mm01),. 4-me~thoxybenzylhydroxylamile (7.368g, 48.lmmol) and mnethanol (25OmL). Heat at reflux: for 16 hours, evaporate the solvent invacuo and add aqueous potassium hydrogen carbonate. Extract with a mixture of 35 ethyl acetate/cyclohexane wash- the combined organic WO 93/05781 PCT/US92/06707 extracts with water and acu~ous sodium chloride. Dry (MgSO 4 evaporate the solvent invczcuo and purify by silica gel chromatography (6:1 cyclohexane/ethyl acetate) to give the title compound as white crystals (16.4g n;±P 59- 62 0

C.

IR (film from CHC1 3 Vmax 2872, 1514, 1454, 2.250, 1136, 1118, 1028, 738, 698 cm- 1 1H NMR (CDCl 3 6 7.35-7.15 (mn, 17H), 6.89 2H, J=8.7Hz), 4.73 1H, J=12.QHz), 4.57 1H, J=12.OHz), 4.55 (d, 1H, J=12.OHz), 4.52 1H, J=12.OHz), 4.38 1H, J=12.3Hz), 4.26 1H, J=12.lHz), 4.28-4.17 (mn, 2H), 3.97 1H, >q-ll.9Hz), 3.95-3.86 (mn, 2H), 3.80 3H), 3.66 1H, J=12.4Hz), 3.65 (dd, 1H, J=5.0, 1.0Hz), 3.47 (d, 1H, J=8.5Hz), 3.25 (qd, 1H, J=8.0, MS 580 552 444, 121; A [a]20 -64.90 1.07, CHCl 3

D

Anal. Calcd for C 3 5

H

3 7 N0 5 C, 76.20; N, 2.54; C, 75.82; H, 6.89; N, 2.38.

Ste-o b: rlS-(lIa,2a,3cx,4B,,5B) 1-[2-(Hydroxvmethvl)-3,4,5- (Dhenvlmethoxy)cyclopentyl (4-methoxyrhenvl)methyllcarbamic acid, 1,l-dimethvlethyl ester Dissolve [3S(aB5,a6a]hxhdol[4 A methoxyphenyl)methyl]-4,5,6-tris(phenylmethoxy)-lH- -cyclopenttc~isoxazole,(4.24g, 7.69mmol) in 6:1 acetic acid/water (42mL) and add activated zinc dust (1.80g, 27.Snunol).' Heat at 50-55 0 C for 105 minutes. Evaporate the solvent in vacua, dI 1u-1e the residue with water and decant the clear aqueous layer from the residual zinc. Wash the zinc with water, aqueous potassium hydroxide and ethyl acetate. Combine all washings and decantates, separate the organic phase and extract the aqueous phase with a mixture of ethyl acetate/cyclohexane Wash the combined WO 93/05781 PCT/US92/06707 -21organic phases with aqueous potassium hydroxide, dilute ammoniumnhydroxide and aqueous sodium chloride. Dry (MgSO4) and evaporate the solvent invacuo to give [iS- (la,2a,3a,4B,5B)]-[2-(hydroxymethyl)-3,4,5- (phenylmethoxy)cyclopetyl] (4-iethoxyphenyl)methylane.

Dissolve [lS-(la,2a,3a,4B,58)]-[2-(hydroxymethyl)-3,4,5- (phenylmethoxy)cyclopetyll[(4-methoxyphenyl)methylaifle (7.69imol) in tetrahydrofurani(75mL) and add di-tert-butyl dicarbonate (3.OmL, 13mmol). Heat at reflux overnight, add additional di-tert-butyl dicarbonate (0.75mL) and heat at reflux fbr 3-yhours. Evaporate the solvent invacuo and purify by silica gel chromatography (60/40 cyclohexane/ethyl acetate) to give the title compound as a viscous oil (4.711g, 94%).

IR (film from CHC1 3 Vmax 3462, 2932, 1688, 1514, 1454, 1366, 1248, 1170, 1124, 1104, 1030, 752, 698 cm- 1 1H NMR (CDC13) 6 7.35-7.22 13H), 7.18-7.05 4H), 6.75 2H, J=8.3Hz), 4.85-4.44 5H), 4.31-3.75 3.7-3.55 311), 3.68 3H), 2.85-2.7 2H), 1.48 1.42 and 1.40 (2s, 9H); MS 654 554 (100), 121; [a] 2 0 +45.08 (c 1.11, CHC13)

D

Anal. Calcd for C 40

H

4 7

N

7 C, 73.48; H, 7.25; N, 2.14.

Found: C, 73.61; B, 7.50; N, 1.91.

SteD c: [lS-(la,2a,3a,4B,5B) ]-[2-Forryl-3,4,5tris(phenlmethoxyj.cvclopentyit 4-mnethoxyDhenyl )methyl]carbamic acid, l,l-dimethvlethyl ester Dissolve [lS-(la,2a,3a,4B,5B) -[2-(hydroxymethyl)-3,4,5- (phenylmethoxy)cyclopentyl) (4-ethoxyphenyl)methylbcarbamic acid, 1,1-dimethylethyl ester (160.6mg, 0.246mmol) in methylene chloride (6iL) and add Dess-Martin periodinane WO 93/05781 PCT/US92/06707 -22- (207mg, O.4B8mxnol). Stir for 15 minutes, pour into a mixture or ethyl ether/water containing potassium hydrogen carbonate (2.61g, 26nunol) and sodium thiosulfate (1.2g, 7.6mmol). Stir until both layers become clear, seoarate the organic phase and wash with aqueous sodium chloride.

Dry.(MgSO 4 and evaporate the solvent invacuo to give the title compound as a colorless oil (158mg).

1H NMR (CDCl 3 6 9.77 and 9.62 (2s in 8:3 ratio, 1H), 7.4- 7.2 (in, 13H), 7.19-7.10 (in, 6.80 2H), 4.63-3.9 (in, 12H), 3.74 3H), 3.14 (in, 1H), 1.44 9H).

Step d: (lS-(lh,2B,3a,4B,5B)]-{2-Formvl-3,4,5tris(phenvlmethoxy)cvclopentyll [(4-methoxyrhenvl)methyl]carbainic acid, 1,1-dimethylethyl ester Dissolve [lS-(la,2a,3a,4B,5B) )-[2-formyl-3,4,5tris(phenylmet'hoxy)cyclopen.tyl][t(4-mr,,thoxyphenyl)methyl]carbamic acid., 1,1-dimethylethyl ester (2.7g, 4.2Srnrol) in methylene chloride (5OmL). Cool to -78 0 C, place under nitrogen atmosphere and add a solution of 1,8diazabicyclo[5.4.0)undec-7-ene (0.195m!,1,.30mmxol) in methylene chloride (0.3mL). Stir for 2 hours, quench at -78 0 C with acetic acid (l0OUiL, l.75mxnol). Parttition between ethyl ether and water, separate the organic phase, dry (MgSO4) and evaporate the solvent inv.acuo to give the title compound as a colorless oil (2.70g, 1H NMR (CDCl 3 6 9.53 (bs, Ill), 7.39-7.19 (in, 15H), 7.14 (d, 2H, J=8.7Hz), 6.77 2H, J-8.7Hz), 4.74-3.75 (mn, 12H) 3.73 3.0-2.7 (bin, 1.45 9H); MS 652 596, 552, 488, 444, 121, 91 (100).

Step e: t1R,2SL5R,4Se5Rj-2-[ dimethylethoxy)carbonvllf(4-methoxvphenyl)methvllaminol-B- WO 93/05781 PCTr/US92/06707 -23hyd roxv- 3, 4, 5-t r is phe nvlme -hoxy cvcloue ntan epr o )a noc acid, ethyl ezter Place lithium hexamethyldisilazide (9.9OmL of a l.OM solution in tetrahydrofuran, 9.9mmol) under a nitrogen atmosphere and cool to -78 0 C. Add, by dropwise addition, ethyl acetate (l.OlmL, 10.3rnmol) arid stir for 15 minutes.

Add, by dropwise addition, a solution of [lStris(phenylmethoxy)cyclopentyl (4-methoxyphenyl)rnethyljcarbamic acid, 1,1-dimethylethyl ester (2.70g, 4.l4mmol) in anhydrous tetrahydrofuran (25mL) and stir for 30 minutes at -78*C. Quench at -78*C with acetic acid, dilute with a mixture of ethyl ether/water and separate the organic phase. Wash the organic phase with aqueous sodium chlor ide, dry (MgSO 4 evaporate th~e solvent in vacuo and purify by silica gel chromatography (77:23 cyclohexane/ethyl acetate) to give the title compound (2-396g, 78.3%).

Anal. Calcd for C 4 4

E

3 i' 3 N0 9 C, 71.43; H, 7.22; N, 1.89.

Found: C, 71.60: H, 7.3.6; N, 1.77.

Ster f: [lR,2S,3R,4S,5R]-2-zU(lfl- ,Dimethylethoxy) carbonyl )amino]-B--hvdroxv-3 tris(p~henvlmethoxy)cvclooentanerornanoic acid, ethyl ester Dissolve [lR,2S,3R,4S,5R]-2-[[ (1,1diLmethylethoxy)carbonyl) (4-rethoxyphenyl)methyllamino]hydroxy'-3,4,5-tris(phenylmethoxy)-cyclopentanepropanoic acid, ethyl ester (2.018g, 2.73mmol) in a 4:1 mixture of acetonitrile/water (75mL) and cool to 0 0 OC -with an ice bath.

Add ceric ammnonium nitrate (4.23g, 7.72mmol) and stir for 1 hour. Pour into a mixture of water/ethyl acetate containing sodium chloride, separate the organic phase, wash with dilute sodium hydrogen carbonate and aqueous sodium chloride. Dry (MgS04), evaporate the solvent invacuG i WO 93/05781 PCT/US92/06707 -24and subject to Kugelrohr distillation to remove 9methoxybenzaldehyde (94 0 C high vacuum) to give the title compound remaining as a white solid which is recrystallized from cyclohexane.

1H NMR (CDC1 3 6 7.39-7.25 15H), 4.70-4.43 7H), 4.2- 3.96 5H), 3.85-3.72 2.5H), 3.58 (bs, 0.5H), 2.62- 2.47 2H), 1.82-1.75 1H), 1.46 9H), 1.25 and 1.24 (2t, 3H, J-7.1Hz); MS 620 564, 548, 521, 520 (100), 91; Exact Mass Calcd for C 36

H

46 N0 8 620.3223; Found: 620.3187.

Anal. Calcd for C 36

H

4 sN0 8 O: C, 69.77; H, 7.32; N, 2.26.

Found: C, 69.52; H, 7.74; N, 2.15.

SteD a: [1R,2S,3R,4S,5R -2-amino-B-hydroxy-3,4,5tris(phenylmethoxy)cyclopentaneurooanoic acid, ethyl ester Dissolve [lR,2S,3R,4S,5R]-2-[[(1,1dimethylethoxy)carbonyl]amino]-B-hydroxy-3,4,5tris(phenylmethoxy)cyclopentanepropanoic acid, ethyl ester (160mg, 0.258mmol) in ethyl ether (20mL) and cool to 0-50C with an ice bath. Bubble hydrogen chloride gas into the solution for 25-30 minutes. Remove the ice bath and evaporate the ethyl ether to 1/2 volume with a stream of nitrogen. Add fresh ethyl ether and carefully add aqueous sodium hydrogen carbonate. Separate the organic phase and extract the aqeuous phase with ethyl acetate. Combine the organic phases, wash with aqueous sodium chloride, dry (MgSO 4 and evaporate the solvent invacuo to give the title compound as a mixture of epimers (140mg).

IR (KBr) vmax 3406, 3358, 1716, 1454, 1366, 1354, 1284,.

1188, 1146, 1116, 1096, 1072, 1052, 1028, 734, 694 cm- 1 MS 560 548 520 100).

WO 93/05781 PCI'/US92/06707 Anal. Calcd for C 3 jH 37 N0 6 C, 71.65; H, 7.18; N, 2.70.

Found: C, 71.58; H, 7.49; N, 2.59.

Step* h: 4aR ,5R, 6S, 7R, 7aS ]-Octahydro-4-hvdroxy-5, 6,7_ tri-!1 (phenvlmethoxy) -2H-1-Dvr indin-2-ole Dissolve tlRe2S,3R,4S,5R]-2-amino-V-hydroxy-,3,4,5t I tris(phenylinethoxy)cyclopentalepropaloic acid, ethyl ester (140mg, O.2E9mmol) in methL~ol (l5mL) and add a catalytic amount of sodium methoxide. Heat at reflux under a nitrogen atmosphere for 90 minutes, add additional sodium V methoxide and continue heating at reflux for 2-1 hours.

Evaporate the solvent invacuo and partition between ethyl ether and aqueous ammonium chloride. Separate the organic phase, wash with acueuos sodium chloride, dry (MgSO 4 :nd evaporate the solvent inuacuo to give the title compound as a yellow, oil whi ch solidi fies on standing (122mg).

IH NMR (C.DC1 3 6 7. 4-7. 28 (in, 15H) 5.94 and 5. 34 (2s in 1: 2 ratio, 1H) 4.73-4. 51 (in, 5H) 4. 45 0. 7H, J=11. 5Hz) 4.44' 0.3H, J-ll.8Hz), 4.23 0.3H), 4.06-3.88 (mn, 3H), 3.82-3.73 (in, 1H), 3.45 (dd, 0.7H, J=11.6, 0 .0 Hz, 2.85 (dd, 0.H ~78 .7(dd, 0.3H, J=18.5, 4.2Hz), 2.46 (dd, 0.3H, J=18.6, 1.5Hz), 2.37 (bs, 0.7H), 2.30 (dd, 0-7Ri, J=18.0, 9.3Hz), 2.08 (bs, 0.3H), 1.80-1.64 (mn, 1H).

Step i: f4S-(4a,4aB,5B,6ci,7a,7aa) ]-Octahydro-5,6,7tris(phenylinethoxy)-lH-1-pyri'ndin-4-ol of structure and 4R-(4a,4aa, 5a,6B,,7B,7aB,) I-octahydro-5,6,77 tris(phenylmethoxy)-lH-1-pyrindin-4-ol (l0b) Add (4aR,5R,6S,7R,7aLS]-octahydro-4-hydroxy-5,6,7tris(phenylmethoxy)-2H-l-pyridi-2-one (884mg, 1.87wimol) to a solution of lithium aluminum hydride (2.5mL of a 1M solution in tvetrahydrofuran) and place under ia nitrogen WO 93/05781 PCr/US92/06707 -26atmosphere. Heat at reflux for 2 hours, cool and pour into dilute aaueous sodium hydroxide. Extract with a mixture of ethyl ether/ethyl acetate wash with aqueous sodium chloride and dry (MgSO4). Evaporatce the solvent invacuo to give a mixture of the title compounds as a colorless oil j (790mg, 92%).

Separate and purify by silica gel chromatography (8-10% methanol/ethyl acetate) to give: 4S isomer (more polar) (203mg, mD 85.5-91.51C.

11' (KBr) Vmax 3534, 2888, 1454, 1352, 1122, 1110, 1088, 10,2, 1030, 740, 696 cm- 1 1H NMR (CDCl 3 6 7.39-7.25 (in, 151F), 4.67 1K, J=ll.7K1z), 4.62 2H), 4.60 1H, J=1l.7Hz), 4.53 1H, J, .11.7Hz), 4.48 1K, J=11.6Hz), 4.15 (in, 1H), 3.96-3.91 (mn, 2H), 3.71 (dd, 1H, J=9.9, 6.2Hz), 3.11 (dd, 1K, J=12.0, 9.9Hz), 2.92-2.74 (in, 2H), 2.06 (bs, 2H), 1.68-1.6 (in, 2K), 1.52 (mn, 1H, J=12.0, 9.2, 2.2Ht); MS (in/z) 500 488 460 100); 2 1 +83.70 (c 1.0, CHCl 3

D

4Anal. Calcd for C 29

H

3 3 N0 4 C, 75.79; H, 7.24; N, 3.05; Found: C, 75.62; H, 7.34; N, 2.99.

254R isomer (less 7.2olar) (424mg, in 110-121C (softens at 105 0 C) IR (KBr) Vmax 3396, 2894, 1140, 1104,' 1076, 1028, 740, 696 cm- 1 KI NMR (CDC1 3 6 7.37-7.25 (in, 15K), 4.68 1H, 4.66 1H, J=l1.5Hz), 4.59 2H), 4.56 2H, 3.95 (dd, 1H, J=6.9, 2.8Hz), 3.87 (dd, 1H, 2.8Hz), 3.81 (dd, 1H, J=9.6, 6.9Hz), 3.54 (td, 1H, J=10.2, 4.6Hz), 3.06 (ddd, 1H, J=12.2, 4.3, 2.2Hz), 2.71 WO 93/05781 PCT/US92/06707 -27- (dd, 1H, J=11.3, 9.9Hz), 2.55 (td, IH, J=12.5, 2.6Hz), 2.39 (bs, 2H), 1.93. 1H, J=12.7Hz) 1.44-1 .29 (mn, 2H); MS 500 488 460 100), 442, 352; (a]21 +45.9- 1.03, CHCl: 3

I).

D

Anal. Calcd for C 2 9

H

3 3 N0 4 C, 75.79; H, 7.24; N, 3.05.

Found: C, 75.53; Ri, 7.49; N, 2.90.

Step [4R-(4a~,4ac,5a,6B,76,7aB) ]-Octahydro-lH-1- Dyvrindine-4 ,5,6 ,7-tetrolohydrochloride Dissolve..[4R-(4a,4aa,5a,6B,7B,7aB)]-oct-ahydro-5, 6 7 tris(phernylmethoxy)-lH-1-pyrildin-4-ol (414mg, 0.O91mnol) in acetic acid (lOmL) and add palladium black (77mg).

in a Parr hydrogenation apparatus for 5 days. Filter through filter aid, rinse with acetic acid and water, and evaporate the solvent invacuo to give an amber oil (289mg).

Purify by ion exchange chromatography ([AG 50W-X8 (Bio- Rad)] (0.lN -0.5N hydrochloric acid) to give the title compound as a white foam (123mg, .lH NMR (D 2 0) 6 4.19 (mn, 1H), 3.96-3.86 (mn, 3H1), 3.57 (ddd, 1H, J=13.3, 4.8, 2.bHz), 3.09 (td, 1H, J=13.6, 3.4Hz), 3.02 (dd, 1H, J=12.4, 9.7Hz), 2.26-2.17 (in, 1H), 1.73-1.57 (mn, 2H).

WO 93/ 05781 PCT/US92/06707 28- Examvole 2 [4S-(4a,4a'3,5B,6a,7a,7aa))--octahydro-lH-1-Dyrindine- 4,5,6, 7-tetrolehydrochloride--MDL-102 ,022A

OH

HO b14 *HCI HO 't

H

Dissolve [4S-(4a,4aa,5$,6a,7a,7aa) I-octahydro-5,6,7tris(phenylrnethoxy)-lH-l-pyrindin-4-ol (173mg, 0.376mmo1) in acetic acid (lOmL) and add palladium black Shake in a Parr hydrogenation apparatus for 4 days. Filter through filter aid, rinse with acetic acid and water, and evaporate the *solvent invacuo to give an amber oil (289mg).

Purify by ion, exchange chromatography ([AG 50W-X8 (Bio- Rad)] (water, 0.1N -0.5N hydrochloric acid) to give the title compound as a white crystalline solid (63mg, mp 217-219 0

C.

I.H NM. (D 2 0) 6 4.31 (in, 1H, J=4.8Hz), 4.15 (dd, 1H, J=9.2, 8.1Hz), 3.95 (dd, lE, J=7.9, 4.3Hz), 3.84 (dd, 1H, J=10.0, 4.3Hz), 3.40 (ddd, 1H, J=13.0, 4.9, 1.6Hz), 3.28-3.17 (in, 2H), 2.11-2.01 (in, 1H), 1.96-1.74 (in, 3H).

354 ,i dllUII~C g IPIl*(- WO 93/05781 PCI/US92/06707 -29- Ex:ample 3 E4R-(4a,4aa,5a,6B,7B,7aB)]-Octahydro-lH-1-methvl-lpyrindine-4,5,6,7-tetrol

OH

HO

HON

HO

10 CH3 Optional Step 12: [4R-(4a,4aa,5a,6B,7B,7aB)1-Octahvdro-lmethvl-5,6,7-tris(phenvlmethoxV)-lH-l-Dyrindin- 4 -cI Dissolve [4R-(4a,4aa,5a,6B,7B,7aB) 3-octahydro-5,6,7tris(phenylmethoxy)-H-l-pyrindi-4-ol (2.30g, 5mmoi) in methanol (distilled from Mg) (50mL) and add formaldehyde (0.405 mL of a 37% solution in water, 5mmol), sodium cyanoborohydride (0.62g, 5mmol) and 1 drop of 1% bromocresol green in ethanol. Maintain the pH of the reaction with 1N hydrochloric acid in methanol until the indicator no longer changes. Evaporate the solvent invacuo and partition the residue between 1N sodium hydroxide and ethyl acetate (100mL). Separate the organic phase, dry' (MgS0 4 and evaporate the solvent invacuo to give the title compound.

SteD k2: [4R-(4o.,4aaQa,6B,7B,7aB)1-Octahydro-l-methyl-lH- 1-pyrindine-4,5,6,7-tetrol Dissolve [4R-(4a,4aa,5a,6B,7B,7aB) ]-octahydro- ethyl- 5,6,7-trisphenylmethoxy)-lH-i-pyrindin- 4 'l (43mg, 0.O91mmol) in acetic acid (lOmL) and add palladium black (77mg). Shake in a Parr hydrogenation apparatus for days. Filter through filter aid,*rinse with acetic acid and water, and evaporate the solvent invccuo. Purify by ion J- I-a- WO 93/05781 PCT/US92/06707 exchange chromatography ((AG 50W-X8 (Bio-Rad)) to give the title compound.

Example 4 r4R-(4a,4aa,5a,6B,7B,7aB)1-Octahvdro-lF-l-benzyl-lpyrindine-4,5,6,7-tetro]

OH

HO

'N

HO

A 15 Oitional Step j2: [4R-(4a,4aa,5a,6B,7B,7aB)-Otahvd ro-l benzvl-5,6,7-tris(phenvlmethoxy)-lH-l-DVrindin- 4 -ol Dissolve [4R-(4a,4aa,5a,6B,7B,7aB)]-octahydro-5,6,7tris(phenylmethoxy)4EH-l-pyrindi-4-ol 12.30g, 5mmol) in methanol (distilled from Mg) (5OmL) and add benzaldehyde (531mg, 5mmol), sodium.cyanoborohydride (0.62g, 5mmol) and 1 drop of 1% bromocresol green in ethanol. Maintain the pH of the reaction with 1N hydrochloric acid in methanol until the indicator no longer changes. Evaporate the solvent in vacuo and partition the residue between LN sodium hydroxide (5OmL) and ethyl acetate (100mL). Separate the organic phase, dry (MgSO4) and evaporate the solvent invacuo to give the title compound.

SteD k 2 [4R-(4a,4aa,5a,6B,7B,7aB)-Octahvdro-lH-l-benzvll-pvrindine-4,5,6,7-tetrol Dissolve [4]-(4a,4aa,5a,68,7B,7a$)]-octahydro-l-benzyl- 5,6,7-tris(phenylmethoxy)-lH-l-pyindin-4ol 0.09lmmol) in acetic acid (lOmL) and add palladium black (77mg). Shake in a Parr hydrogenation apparatus for days. Filter through filter aid, rinse with acetic acid WO 93/05781 PCI'/US92/06707 -31and water, and evaporate the solvent invacuo. Purify by ion exchange chromatography ([AG 50W-X8 (Bio-Rad)] to give the title compound.

Example [4R-(4a,4aa,5a,6B,7B,7aB)]-Octahvdro-lH-l-(1,3dihvdroxvToro-2-vl )-l-Tvrindine-4, 5,6 ,7-tetrol

OH

HO

HO HO I CHC2OH) 2 Optional Steip 12: (4R-(4a,4aa,50,68,7B,7aB)1-Octahvdro-l- 3-dihydroxy-oroo-2-vl 7-tris (Dhenvlmethoxv) -l-lpyrindin-4-ol Dissolve [4R-(4cx,4aa,5a,6B,7B,7aB) ]-octahydro-5,6,7tris(phenylmethoxy)-lH-l-pyrindin-4-ol (1.22g, 2.65mnxol) in methanol (l7mL) and add 1,3-dihydr-oxyacetone dimer (497mg, 2.76mxnol) and sodium cyanoborohydride (202g, 3.2lminol).

Stir atL room temerature for 24 hours, evaporate the solvent invacuo and partition the residue between aqueous potassium hydroxide (50mL) and ethyl acetate (lO0mL). Separate the organic phase, dry (MgSO 4 evaporate the solvent invacuo and purify by silica gel chromatography to give the title compound Ste [4R-(4a,4aa,5c±,68,.78,7a8) ]-Octahydro-lH-1-(l,3dihydroxy]prop-2-vl)2-pvrindine-4 7-tetrol Dissolve (4R-,(4a,4aa,5ci,6B,7B,7aB) I-octahydro-l-(l,3dihydroxyprop-2-yl)-5,6,7-tris(phenylmethoxy)i!H-1 pyrindin-4-ol (50mg, 0.O91mmol) in acetic acid (lOmL) and add palladium black (77mg). Shake in a Parr hydrogenation WO 93/05781 PCr/US92/06707 -32apparatus for 5 days. Filter through filter aid, rinse with acetic acid and water, and evaporate the solvent in vacuo. Purify'by ion exchange chromatography ([AG 50W-X8 (Bio-Rad)) to give the title compound.

ExampDle 6 f 4

R-(

4 a,4aa,5o.,68,7B,7aB)]-Octahydro-lH-1-(6-deoxvi-l-0methvl-mannosyl )-l-o~vrindine-4,5,6, 7-tetro.

OH

HO,-

HO OH 0

HO

OHR

Optional Step j2: 4 R-(4o.,4aa,5ca,6B,7B,7a'B)]-Octahvdro-l- (-deoxy-1-0-methvl-mannosyl)-5,6,7-tris(phenvlmethoxv)-lHil-nvrindin-4-oi Mix potassium carbonate (96.2mg, 0.696nxol), 6-bromo-6deoxy-l-O-methyl-2,3,4-tris(phenylmethoxy)mannose (264.3mg, 0.50lmmol) [.4R-(4cL,4aa,5ca,6B,7B,7aB) I-octahydro-5,6,7tris(phenylmethoxy)-lH-l-pyrindin-4-ol (229mg, 0.498nmol) and n-butanol (6mL). Heat at reflux under a nitrogen atmosphere for 8 days. Cool, pour into a mixture of ethyl acetate/water and separate the organic phase. Wash the organic phase with aqueous sodium chloride, dry (MgSO 4 and evaporate the solvent in vacuo. Purify by silica gel chromatography to give the title compound.

Sten k2: t4R-(4a,4aa,5a,6B,7B,7aB) ]-Octahydro-lH-1-(6deoxy-1-0-methyl-mannosyl)-l-pyrindine-4,5,6,7-tetro1 I\ ~r~au~ara~ WO 93/05781 PCT/US92/06707 -33- Dissolve 4

R-(

4 a,4a,5a,6B,7B,7aB)]-octahydro-l-(6-deoxy-l- O-methyl-mannosyl)-5,6,7-trls(phenylmethoxy)-lH-1-pyrindin- 4-ol (82mg, 0.091mmol) in acetic acid (10mL) and add palladium black (77mg). Shake in a Parr hydrogenation apparatus for 5 days. Filter through filter aid, rinse with acetic acid and water, and evaporate the solvent in vacuo. Purify by ion exchange chromatography ([AG 50W-X8 (Bio-Rad)] to give the title compound.

The compounds of this invention are alpha-mannosidase inhibitors, immunomodulatory agents, chemoprotective agents, radioprotective agents and antimetastatic agents.

In practicing the method of this invention, an effective amount of a compound of this invention is that amount required to inhibit Mannosidase II and thus to elicit a chemo- or radioprotective, immunostimulatory, or antimetastatic effect. Immunostimulatory agents are desirable in those instances where the immune system of the patient has been compromised, such as in those patients infected with HIV, the causative agent in AIDS and ARC, as well as patients undergoing bone marrow transplants and in patients having various cancers. The compounds of this invention can also be used to prevent or to treat metastasis of tumors. In addition, the compounds of this invention can be used as chemo- and radioprotective agents by virtue of the ability of these compounds to reduce myelosupDression with resultant leukopenia and by virtue of the compounds to stimulate hematopoietic activity.

The specific dosage for the treatment of.any specific patient in need of chemoprotective, radioprotective, immunostimulant or antimetastatic therapy will depend upon such factors as size, type,.and age of the patient as well as the severity of the disease state, all, of which are WO 93/05781 PCT/US92/06707 -34factors normally familar to and considered by the attending diagnostitian treating the patient. Generally, the compounds are to be administered orally at a dose of from 0.2 to 20 mg/kg of patient body weight per day, with a dose of from 0.5 to 5 mg/kg beihg preferred. The compounds preferably are to be administered orally at mealtimes in single or multiple unit doses containing from 25 mg to 250 mg of the chosen compound.

In practicing the method of this invention, the active ingredient is preferably incorporated in a composition comprising a pharmaceutical carrier and from about 5 to about percent by.weight of a compound of the invention or a pharmaceutically-acceptable salt thereof. The term "pharmaceutical carrier" refers to known pharmaceutical excipients useful in formulating pharmaceutically active compounds for internal administration to animals, and which are substantially non-toxic and non-sensitizing under conditions of use. The compositions can be.prepared by known techniques for the preparation of tablets, capsules, elixirs, syrups, emulsions, dispersions and wettable and effervescent powders, and can contain suitable excipients known to be useful in the preparation of the particular type of composition desired.

The preferred route of administration is oral. For oral administration the formula 1 compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, troches, lozenges, melts, powders, solutions, suspensions, or emulsions. The solid unit dosage forms can be a capsule which can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers such as lactose, sucrose, calcium phosphate, and cornstarch. In another embodiment the compounds of this invention can be WO 93/05781 PCT/US92/06707 tableted with conventional tablet bases such as lactose, sucrose, and cornstarch in combination with binders such as acacia, cornstarch, or gelatin, disintegrating agents intended to assist the break-up and dissolution of the tablet following administration such as potat6 starch, alginic acid, corn starch, and guar gum, lubricants intended to improve the flow of tablet granulations and to prevent the adhesion of tablet material to the surfaces of the tablet dies and punches, for example, talc, stearic acidi or magnesium, calcium, or zinc stearate, dyes, coloring agents, and flavoring agents intended to enhance the aesthetic qualities of the tablets and make them more acceptable to the patient. Suitable excipients for use in oral liquid dosage forms include diluents such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent.

The formula 1 compounds of this invention may also be administered parenterally, that is, subcutaneously, intravenously, intramuscularly, or interperitoneally, as injectable dosages of the compound in a physiologically acceptable diluent with a pharmaceutical carrier which can be a sterile liquid or mixture of liquids such as water, saline, aqueous dextrose and related sugar solutions, an alcohol such as ethanol, isopropanol, or hexadecyl alcohol, glycols such as propylene glycol or polyethylene glycol, glycerol ketals such as 2,2-dimethyl-l,3-dioxolane-4methanol, ethers such as polyethyleneglycol 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant such as a soap or a detergent, suspending agent such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose,

I'

I.

i It ii I ~II. WO 93/05781 PCT/US92/06707 -3F;or carboxymethylcellulose, or emulsifying agent and other pharmaceutically acceptable adjuvants. Illustrative of oils which can be used in the parenteral formulations of this invention are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum, and mineral cil. Suitable fatty acids include oleic acid, stearic acid, and isostearic acid. Suitable fatty acid esters are, for example, ethyl oleate and 10 isopropyl myristate. Suitable soaps include fatty alkali metal, ammonium, and triethanolamine salts and suitable detergents include cationic detergents, for example, dimethyl dialkyl ammonium halides, alkyl pyridinium halides; anionic detergents, for example, alkyl, aryl, and 15 olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulf-ates, and sulfosuccinates: i. nionic detergents, for example, fatty amine oxides, i ty acid alkanolamides, and polyoxyethylenepolypropylene copolymers; and amphoteric detergents, for example, alkyl beta-aminopropionates, and 2-alkylimidazoline quarternary ammonium salts, as well as mixtures. The parenteral compositions of this invention will typically contain from about 0.5 to about 25% by weight of the formula 1 compound in solution.

Preservatives and buffers may also be used advantageously.

In order to minimize or eliminate irritation at the site of injection, such compositions may contain a non-ionic surfactant having a hydrophile-lipophile balance (HLB) of from about 12 to about 17'. The quantity of surfactant in such formulations ranges from about 5 to about 15% by weight. The surfactant can be a single component having the above HLB or can be a mixture of two or more components having the desired HLB. Illustrative of surfactants used in parenteral formulations are the class of polyethylene sorbitan fatty acid esters, for example, sorbitan monooleate and the high molecular weight adducts of 11111~CI1- i -i WO 93/05781 PC/US92/06707 -37ethylene oxide with a hydrcphobic base, formed by the condensation of propylene oxide with propylene glycol.

The compounds of this invention can also be administered topically. This can be accomplished by simply preparing a solution of the compound to be administered, preferably using a solvet known to promote transdermal absorption such as ethanol or dimethyl sulfoxide (DMSO) with or without other excipients. Preferably topical administration will be accomplished using a patch either of the reservoir and porous membrane type or of a solid matrix variety.

Some suitable transdermal devices are described in U.S.

Pat. Nos. 3,742,951, 3,797,494, 3,996,934, and 4,031,894.

These devices generally contain a backing member which defines one of its face surfaces, an active agent permeable adhesive layer defining the other face surface and at least one reservoir containing the active agent interposed between the face surfaces. Alternatively, the active agent may be contained in a plurality of microcapsules distributed throughout the permeable adhesive layer. In either case, the active agent is delivered continuously from the reservoir or microcapsules through a membrane into the active agent permeable adhesive, which is in contact with the skin or mucosa of the recipient. If the active agent is absorbed through the skin, a controlled and predetermined flow of the active agent is administered to the recipient. In the case of microcapsules, the encapsulating agent may also function as the membrane.

In another device for transdermally administering the compounds in accordance with the present invention, the pharmaceutically active compound is contained in a matrix from which it is delivered in the desired gradual, constant 'i WO 93/05781 PCI7US92/06707 -38and controlled rate. The matrix is permeable to the release of the compound through diffusion or microporous flow. The release is rate controlling. Such a system, which requires no membrane is described in U.S. Pat. No.

3,921,636. At least two types of release are possible in these systems. Release by diffusion occurs when the matrix is non-porous. The pharmaceutically effective compound dissolves in and diffuses through the matrix itself.

Release by microporous flow occurs when the pharmaceutically effective compound is transported through a liquid phase in the pores of the matrix.

i

Claims (12)

1. A compound of the formula OH HO,,, HO N HO R wherein R is a hydrogen, a (C-C 6 )alkyl optionally substituted with one or two hydroxy groups, a glycosyl group, or a group of the formula -(CH 2 )N-Ar wherein n is an intege" of from 1 to 4 and Ar is a phenyl group optionally substituted with one or two groups selected from (Cl-C 4 )alkyl, (C 1 -C 4 )alkoxy, F, Cl, Br, I, amino, mono(C-C 4 )alkylamino, or di(Cl-C 4 )alkylamino, or a pharmaceutically acceptable salt thereof.

2. A compound of claim 1 wherein R is a hydrogen, methyl, 1,3- dihydroxypiop-2-yl, benzyl or mannosyl group.

3. A compound of claim 1 or claim 2 wherein the 4-hydroxy group is of the alpha-configuration.

4. A compound of claim 1 or claim 2 wherein the 4-hydroxy group is of the beta-configuration.

A compound according to claim 1 wherein the compound is [4R- (4a,4aa,5a,6p,7p,7ap)]-octahydro-1 H-1-pyrindine-4,5,6,7-tetrol.

6. A compound according to claim 1 wherein the compound is [4S- 20 (4a,4ap,5p,6a,7,7aa)]-octahydro-1 H-1-pyrindine-4,5,6,7-tetrol. S'

7. A method for treating immunodepression in a patient in need thereof which includes administering to the patient a therapeutically effective amount of a compound of any one o' the preceding claims.

8. A method for pr.venting or treating metastasis in a patient in need thereof S 25 which includes administering to the patient a therapeutically effective amount of a compound of any one of claims 1 to 6. !T i -ii i l- I rll

9. A composition including a compound of any one of claims 1 to 6 and a carrier.

A composition according to claim 9 in the form of a pharmaceutical composition wherein the carrier is one or more pharmaceutical carriers.

11. A method for treating chemotherapy and radiotherapy associated myelosuppression and leukopenia in a patient in need thereof which includes administering to the patient an effective amount of a compound according to claims 1 to 6.

12. A compound according to any one of claims 1 to 6 substantially as hereinbefore described with reference to the examples. DATED: 22 February, 1995 PHILLIPS ORMONDE FITZPATRICK Attorneys for: MERRELL DOW PHARMACEUTICALS INC. D r r I I I r I P I zr~ INTERNATIONAL SEARCH REPORT International application No. IPCTIUS92/06707 A. CLASSIFICATION OF SUBJECT MAATTER :A61K 11/44 US CL :511443,70,445 According to International Patent Classification (IPC) or to both national claasification and IPC B. FIELDS SEARCHED Minimum'documentation searched (classification system followed by classification symbols) U.S. 514/299; 536/23; 546/112 Documentation searched other than mrinimumr documentation to the extent that such documents are included in the fields searched Electronic data base consulted daring the international search (name of data base and, where practicable, search terms used) STRUCTURE SEARCH C. DOCUMENTS CONSIDERED TO BE RELEVANT Category's Citation of document, with indication, where appropriate, of the relevant passages Relevant to claimn No, A US,A, 4,031,102 (CURRAN) 21 JUNE 1977. SEE THE ENTIRE DOCUMENT. 1-11 A GB,A, 2 088 860 (SHEPHERD) 16 JUNE 1982 11 Furth er documents are listed in the continuation of Box C. Sec patent family annex. Special categores of cited documns: *T Later document published sfier the international iling date or priority *A dciamedemiqthe oneai sateof te ~date and notin conflict with the application but cited to understand the W doumetelfinnj he eneal o th anwhih i no cosidredprincile or theory Lnderlying the invention to be pan Of Pitnctlr relevaunce E' ealir ocmet ubisedon or after the inetionl fln date considered novel or cannot be considered to involve an inventtve step docmmt which may throw doushb an prior*t claim(s) or which is when the document in taken alone cited to establish the publicion 4ta&e of anose citation or other opwW neecia (a qefiud) Y docuitent of particuliar relevance; the claimed Inventio cinnot be comidered io involve an inventive step when the document is 0, doctameast referring to anoral diockoure use, exhibition or Other combined wRkt one or more other such documents, such comnbination messisbeiiog obvious to a person skille in the an *P doausaaipublissed prior to the kternutioei fiing date b4tt later than doctimal member of the manse patent family die priority dam le eird Date of the actual completion of the international search Date of mailing of the international search report 08 SEPTEMbErft 19922 Name and milfing address of the ISA/ Authorized officer Commisaiocer of Patents Wn Trademarks Box PcT W&.~ngton, D.C. '10231 KI R Fa,:iimile No. NOT APPLICABLE .Telephone No. _(03 308-2351 Form PCTIISA/21O (second sheeX)jty 1992>*